The three myosin isoenzymes, IA, IB and II, of Acanthamoeba castellanii also occur in related amoebae and seem to have different intracellular localizations: myosin IA and IB being preferentially localized at or near the periphery of the cell. Despite their unusual subunit composition, the isolated myosins seem to be the native forms of the molecules. The ATP-site and the phosphorylation-site on the heavy chain of myosin IA can be separated from each other by subtilisin digestion. Subtilisin digestion also produces lower molecular forms of myosin IA whose Mg-ATPase activity can be actin-activated without the phosphorylation that is required for the native enzyme. A highly actin-activatable form of myosin II has been isolated for the first time. It contains about 0.97 mol P/mol of heavy chain and its actin-activated ATPase activity is enhanced 3-4 fold by dephosphorylation to 0.35 mol P/mol of heavy chain. This is the first example of dephosphorylation, rather than phosphorylation, enhancing an actomyosin ATPase. Therefore, in the same cell the ATPase activities of actomyosin IA and IB are enhanced (derepressed) by phosphorylation of their heavy chains and the ATPase activity of actomyosin II is enhanced by dephosphorylation of its heavy chain.